Background: Myofibroblasts (MF) from human granulation tissue derive from perilesional cells and display similarities with fibroblasts (DF) and smooth muscle cells (SMC). Objective: Determine additional markers to assess the phenotype of MF. Methods: MF, DF and SMC derived from human tissues were amplified in culture. Their phenotype and response to various regulatory processes were compared in vitro. Results: MF resemble SMC by their mechanical properties and cytoskeletal proteins. They resemble DF by their biosynthetic phenotype and their response to the regulation operated by the extracellular matrix and mechanical forces. Conclusion: In vitro MF display a phenotype that differentiates them from DF and SMC.